DK1587824T3

DK1587824T3 - PROCEDURE FOR PROCESSING CONCENTRATED ENZYME SOLUTIONS

Info

Publication number: DK1587824T3
Application number: DK04704587.7T
Authority: DK
Inventors: Dieter Baur; Werner Pichler; Wilfried Rähse; Holt Jens Van
Original assignee: Basf Se
Priority date: 2003-01-31
Filing date: 2004-01-23
Publication date: 2015-03-23
Also published as: HK1085747A1; CN1745093A; CN100528895C; WO2004067557A1; DE10304066B4; DE10304066A1; EP1587824B1; US20050266542A1; EP1587824A1; ES2533001T3

Description

The present invention relates to methods for refining concentrated technical enzyme solutions, to the corresponding method products and to compositions based on such solutions, more particularly laundry detergents and cleaning products.

Enzymes, especially those for technical fields of use, are nowadays usually produced by fermentation of microorganisms and are subsequently purified from the media in question. The concentrated enzyme solutions that are obtained via usually a plurality of sequential process steps are often also referred to as "liquid enzyme". Liquid enzyme can be viewed as a purified raw material which either is used in liquid form or -less often - is converted into a dry form and then passed on for appropriate applications.

Important technical fields of use for enzymes, particularly in liquid form, are laundry detergents and cleaning products, which increasingly are also being offered in liquid or gel form. Other fields of use are in cosmetics, for example, with the enzymes, as in laundry detergents and cleaning products, being used as active agents, or in the manufacture and processing of textiles, or in food production, in which case primarily the raw materials are converted to the actual product through use of the enzymes.

Purification or enrichment methods for obtaining concentrated enzyme solutions have been comprehensively described in the prior art. Important objectives in such methods are the removal of the biomass, in other words the constituents especially macromolecular constituents - of the host organisms, the removal of low molecular mass accompaniments and impurities, especially media constituents and metabolites, and the removal of other proteins, especially enzymes. At the same time, the product of interest is to be obtained in as great as possible an amount, purity, and as high as possible an activity. On the other hand, the culture supernatants usually contain factors, often peptides or proteins, whose identity in many cases is as yet unknown, but which ensure stabilization of the enzyme of interest. It is particularly advantageous, therefore, to obtain not a fully pure enzyme solution but rather one which includes a certain proportion of such stabilizing accompaniments.

For the purpose of purification, techniques based on filtration, sedimentation or precipitation are usually employed.

For example, for the removal of the biomass, methods have been developed which are usually to be employed in succession, and are established in the art. Such methods include, for example, separation, microfiltration, and ultrafiltration. Only afterward is it possible, in the sense of the invention, to refer truly to an enzyme concentrate.

Thus, for example, patent application WO 01/37628 A2 reveals a method for recovering biotechnologically produced substances of value from culture solutions and/or fermenter solutions, said method comprising the separation of the water-insoluble solids from the aqueous solution comprising the substances of value, followed by filtration of the resultant solution and concentration of the solution containing substance of value, by means of ultrafiltration. The method is characterized in that the solids removed are subjected to a washing step, with the washing liquid used being the filtrate from the concentration stage.

Still unresolved, however, is the problem that enzyme concentrates removed from the biomass contain in particular the following accompaniments: 1. solids, especially precipitates of irreversibly denatured proteins, including those of the protein of interest; 2. compounds which are colored, usually brown, and which have formed in the pre-fermentation sterilization of the media constituents, particularly of the nitrogen sources (Maillard compounds); and 3. factors which raise the stability of the protein of interest.

Conventional refining techniques are suitable only to an inadequate extent for removing denatured proteins and colored compounds from an enzyme concentrate separated from the biomass; or these techniques, together with these proteins and compounds, also remove a large portion of the stabilizing factors. The consequence of this in each case is unsatisfactory product quality: either the enzyme concentrate has a dark color, contains streaks and/or suspended materials, or even precipitated materials, or, with a light color and a clear solution, it possesses inadequate enzyme stability; the latter can be compensated usually only to a certain degree by addition of (expensive) stabilizing compounds. These disadvantages have consequences in particular for the products into which the concentrate in question is incorporated.

Liquid laundry detergents or cleaning products, for example, ought to contain a very high fraction of active, i.e. stable, enzyme over the storage period. However, water content and stability are in inverse proportion to one another. At the same time, these compositions ought to possess a color which is appealing to the user, and a clear appearance.

Methods for the decolorizing of concentrated enzyme solutions have been described in the prior art. They include precipitation techniques, with organic solvents or polymers, for example, but also include, in particular, the salting-out of the protein of interest with sodium sulfate (described in H. Ruttloff (1994): "Industrielle Enzyme" [Industrial Enzymes]

Behr's Verlag, Hamburg, section 6.3.3.6, pages 376 to 379). Thus, for example, patent US 5405767 reveals certain compounds which are to be added to the protein solution for precipitation, in order to obtain advantageous precipitates. Here, the protein is precipitated, with the accompaniments remaining in the supernatant. However, as a result of the precipitation and resuspension, part of the protein is irreversibly denatured and overall the stability is impaired -not least for reasons including the removal of stabilizing compounds as well. The achievable yield is indicated, in the table on page 378 of the textbook cited, as being around 50%.

Another alternative is the adsorptive purification of the enzymes, by way of an ion exchange resin, for example (H. Ruttloff (1994): "Industrielle Enzyme" [Industrial Enzymes] Behr's Verlag, Hamburg, sections 6.3.3.7 and 6.3.3.8, pages 379 to 396) . Here, the proteins of interest bind to a chromatography material and are subseguently eluted with a different medium. As a result, however, because of denaturing effects and folding effects, poor yields are again usually obtained. Accordingly, for various chromatography methods (apart from affinity chromatography), the table on page 378 reports yield levels of about 60% at most. The specific materials, more particularly the affinity chromatography materials, are generally more highly performing, but are highly sensitive and are costly and inconvenient to produce. These materials are therefore employed predominantly in analysis and in medical technology, but hardly at all in industrial enzyme manufacture.

Thus, for example, patent application WO 89/05863 A1 describes the recovery of extracellular enzymes from the fermentation broth from Bacillus strains. It describes experiments for the removal of cell wall polymers via ion exchange chromatography, especially as part of an amylase preparation procedure. First of all, the solution, containing enzyme and polymer, is applied to the column, which is then rinsed first with buffer and subsequently eluted with an elution medium. In other words, the alpha-amylase for purification initially binds, like the accompaniments, to the chromatography material, and only then, on increase of the ionic strength, is washed down.

The converse approach, namely the deliberate depletion of the impurities from the liquid solution via a support material, has been selected so far only for raw materials for foods. For instance, patent US 5972121 discloses the removal of colorants from sugar solutions via a weakly acidic or weakly basic adsorption chromatography. Additionally, in the handbook "DIAION®. Manual of ion exchange resins and synthetic absorbent, Volume II" from Mitsubishi Kasei Corp. (Tokyo, Japan), 2nd printing, May 1, 1993, pages 93 to 100 describe the decolorizing of sugar by means including different ion exchange chromatography steps connected in series. This converse approach gives rise in principle to a plurality of purification steps, which in detail are each highly selective, in which the impurity which is removable in each case is left on the corresponding material.

An example in patent US 5565348, which is concerned with the recovery of a particular alkaline protease of a bacillus, describes the purification of this enzyme via a purification procedure that consists of numerous steps. These include, in particular, the steps of precipitating the protein by salting out, the taking up of the protein in another medium, and a dialysis, before an ion exchange chromatography is carried out; this is followed in turn by an affinity chromatography, in other words a chromatography step in which the protease binds specifically to the column material. With regard to the ion exchange chromatography step, it is said that the specific protease described did not bond to the specific chromatography material used and was therefore obtained with the break-out. However, this does not appear to be a generalizable teaching for the purification of the enzyme, since no details are given of the column material in question, the concentration of the enzyme is not reported, and the actual purification to remove accompaniments has already taken place in the preceding precipitation, and/or is ensured by the subsequent affinity chromatography. Accordingly, in the prior art to date, there had been no consideration given to the purification of an enzyme, more particularly a protease, by way of a column material to which the protease itself does not bind at all, and especially not to a procedure not involving a precipitation step.

The object was therefore to free enzyme concentrates from solids, particularly from irreversibly denatured proteins, and to decolorize them targetedly in such a way that at the same time extremely high storage stability of the concentrates is maintained.

This object is achieved by methods for refining concentrated enzyme solutions according to claim 1.

In this case there is no precipitation. Instead, the enzyme proteins of interest remain in solution throughout the entire process; for this purpose, as set out below and in the examples, particular concentration ranges are particularly advantageous in respect of the achievable yield.

Step (a) of the method is preceded by conventional methods, described in the prior art, for the production of largely biomass-free, enriched, aqueous enzyme solutions. In general these involve a number of component steps, such as cell digestion, pelletizing of the cell debris, decanting, and optionally further centrifugation steps. In addition, separation, microfiltration, ultrafiltration or sterile filtration procedures (see below), and concentration, in other words removal of the solvent down to a middle concentration range of the enzyme, may already be employed. An enzyme concentration level which can be regarded as optimum here is one which lies in the size range of half of the range identified as the optimal working concentration in the further method (see below). Additionally advantageous is an achievable suspended-materials or solids fraction of below 1% by volume, as may be verified, for example, by a centrifugation with a bench centrifuge for 10 minutes at 7000 g. The solution in question ought, moreover, to have been adjusted to a pH which is compatible for the enzyme and at which it has a positive charge .

Step (a) as well, which is identified as "concentration" in figure 1, is based on techniques which are known per se to the skilled person. For the concentration it is possible, for example, to use a rotary evaporator or a thin film evaporator. It is particularly advantageous in this case if the pH as adjusted beforehand remains largely constant, and the solids fraction of the enzyme concentrate remains very low (see below). Otherwise, there is a disadvantageous increase in the losses in the subsequent step (b).

Step (a) is to be controlled by way of the known techniques (in particular by timely termination of the concentration procedure) in such a way that the enzyme concentrate obtained still just exhibits no (quantitative) protein precipitation. The optimum operating range here must be determined individually for each enzyme, not only in terms of the temperature, pH, and ionic strength but also, in particular, in terms of an optimum enzyme concentration range. This was performed in Examples 1 and 2 of the present application for the alkaline protease from Bacillus lentus and for the a-amylase from Bacillus sp. A 7-7 (DSM 12368) . For the protease under investigation, accordingly, an optimum concentration range of 700 000 to 800 000 HPE/g was found, and for the a-amylase an optimum concentration range of 35 000 to 45 000 TAU/g. Above these levels, there is soon a disproportionate increase in the fraction of precipitated solids, depending on the activity, and this is accompanied by a massively increasing loss of good product. These effects are illustrated for the stated example enzymes by figure 2. A dependency relationship of this kind between the solids fraction and the concentration of protein, especially of enzyme activity, can be expected for virtually all technical enzymes. It must be determined experimentally in each individual case, and the method must be aligned thereto via the conventional concentration methods and, optionally, dilution methods.

As is likewise shown in the examples, it is preferred to stay within this operating range as far as possible throughout the entire process, in order first to operate at high concentration and hence efficiently, but on the other hand to lose as little enzyme as possible through denaturation and precipitation. In this way it has been possible to achieve yields of up to 95% for the overall process.

Step (a) is followed optionally by the deodorization (a') indicated in figure 1. This is addressed later on below.

The removal of the precipitations (solids) formed by concentration, in step (b) , relates in particular to foreign proteins and/or inactive enzymes, particularly in the vicinity of the solubility product. This step is identified in the flow diagram of figure 1 as "Separation". It takes place likewise in accordance with techniques that are known per se, via a separator (see below) , for example, as is also disclosed in the examples for the present patent application.

In the supernatant containing the enzyme of interest, there should now be as little as possible (see below) of suspended materials, in other words solids, which, as already indicated above, can be determined via a bench centrifuge. The reason is that proteins precipitated as solids cannot be brought back into solution through dilution without considerable loss (see above) and only with a massive reduction in concentration. Moreover, like other solids, they impair the subsequent chromatography step, since they can block the column.

Step (c) , with the decolorizing of the largely solids-free supernatant from step b) via a strongly basic anion exchanger (adsorber), constitutes the core of the invention. In this step, the colored accompaniments in particular are adsorbed to the resin, especially the Maillard compounds, whereas the strongly positive charge of the exchanger means that the proteins, which are likewise positively charged under conditions to be selected correspondingly, are not bound on the resin, but are instead obtained with the eluate in a largely clear solution. Step (c) therefore represents a selective removal of the colorants from the concentrate enzyme solution.

The advantage of this method over the methods described in the prior art is that the substances of interest for their value, in other words the enzyme proteins, remain in solution, and so do not have to be denatured and renatured, and therefore are not altered in their three-dimensional structure. Accordingly, they also remain in the phase which is for further processing, and are not discharged from the system. As a result it has been possible to achieve the high yields referred to above.

The predominantly colored substances bound to the resin are subsequently - as indicated in figure 1 by corresponding bold arrows, in other words after emergence of the phase containing substance of value (the good product) and optionally a postfraction, are eluted in a separate step. This is accomplished, for example, using solutions with a high ionic strength, such as concentrated NaCl solutions. Via corresponding counterions, NaOH for example, the anion exchange material can be regenerated. Depending on the chromatography material, other simple salts may be better suited. The fact that this material can be treated with compounds of this kind - which, moreover, are cost-effective - results in sterilization as well as the purification effect. The system is therefore "CIP"-able (for "cleaning in place") .

Following step (c) of the method, the liquid enzyme is largely free from disruptive streaks, precipitates, and colorants. Even with prolonged storage at varying temperature, it remains pale, clear, and bright, and at the same time has a high level of stability. Examples of color values which can be achieved, in accordance with the internationally customer CIE color scale (defined in DIN 5033-3 and DIN 6174), are apparent from the experiments described in the examples of the present patent application.

Step (c) of the method is optionally followed by step (d) , described more comprehensively later on below, where the highly concentrated chromatography product is mixed with solvent. This is also illustrated by figure 1 ("mixture").

This refined concentrated enzyme solution obtained after step (c) and/or (d) , is significantly depleted with respect in particular to the colored impurities, but still contains virtually colorless accompaniments which, on account of their partly stabilizing effect, are very welcome and are not to be/need not be removed from the concentrated enzyme solution. Additional interim steps may be carried out in advance, inserted, added on, or carried out together with the stated steps, depending on the separation task. Examples of three such optional interim steps are given below. A further possibility, for example, is to carry out deliberate removal of further accompaniments from the concentrated enzyme solution by means of one or more additional chromatography steps, in particular using different support materials, which are adequately described in the prior art (see above) . This can take place at any point in time in the method that appears sensible in the specific case, advantageously immediately before or after the chromatography step described under (c) , optionally separated from one another by interim steps such as filtrations or resolubilizations. A change of solvent, which may be performed at various points of the method of the invention, preferably before or in place of step (d) , is evident from patent application DE 19953870 Al, for example. Disclosed therein is a method for producing a low-water-content enzyme preparation comprising an organic solvent, in which an aqueous enzyme preparation is mixed with an organic solvent having a boiling point of more than 100°C and the water is subsequently removed by distillation.

The liquid enzyme obtained by methods of the invention can be further processed or used in a manner known per se. It is particularly important as a raw material for incorporation into laundry detergents or other cleaning products, especially in liquid form. The balance required in accordance with the invention between the clear color of such products and the more than satisfactory stability is illustrated in example 3, the result of which is also shown in figure 3. There it is seen that the product refined in this way is only slightly less stable than the impure enzyme, but in contrast to that enzyme it is virtually colorless, and, on the other hand, it is still very much more stable than a commercial product which has been decolorized conventionally, namely via precipitation.

Set out below are preferred embodiments and further subjects of the invention.

As mentioned above, enriched aqueous enzyme concentrates, freed from the biomass by conventional methods described in the prior art, are introduced into step (a) of the method. In general, a number of component steps are necessary to accomplish this. In preferred methods of the invention, as a final step, immediately before the step (a) in the method, an ultrafiltration is performed, and so an ultrafiltration concentrate is introduced into step (a) according to the invention. A method of this kind is described in WO 01/37628 A2, for example. As a result, comparatively pure enzyme solutions are obtained that are already enriched to a middle concentration value and that have a low solids content (compare example 1).

As mentioned, conventional techniques are used for the concentration procedure of step (a), such as techniques, for example, involving a rotary evaporator or a thin film evaporator; the latter embodiment is preferred.

In another preferred embodiment, step (a) is controlled via the parameters to be set in each case, in particular the duration of the concentration procedure or, optionally, dilution, in such a way as to give an enzyme concentrate which comprises not more than 4 to 20% by weight, preferably not more than 4.5 to 15% by weight, particularly preferably not more than 5 to 10% by weight of dry substance.

In contrast to the unwanted solids described above, the dry substance constitutes the entire solid-substance content of the concentrated enzyme solution, as obtainable, for example, by complete evaporation of the solution. These figures can be determined by techniques that are known per se, such as by drying an aliquot or by absorption measurement and comparison with a calibration plot. The figures specified have emerged as particularly suitable values in the context of the further processing. On the one hand, indeed, the solution should be as highly concentrated as possible, in order to avoid losses; on the other hand, too high a viscosity would lead to difficulties in the constant throughput.

In a further preferred embodiment, in methods according to the invention, a deodorization of the concentrated enzyme solution is carried out subsequent to step (a) in step (a'). Deodorization techniques of this kind, which can be integrated into a continuous operation, are known from the prior art. They are especially preferred when the microorganisms used for manufacturing the enzyme protein are microorganisms which form accompaniments with an unpleasant odor, or excrete proteins which very rapidly break down other constituents.

Step (b) of the method, the removal of solids, which follows (a), or follows the deodorization (a'), is also based on conventional techniques, filtration being an example. Among such techniques, however, preference is given to mechanical separation processes, preferably based on gravitational or centrifugal separation.

Included in this, in particular, are separators, preferably continuously operating separators, which can be integrated into a continuous operation or regime. Particularly preferred among these are those with intermittent discharge of sediment. They are also disclosed by the examples for the present patent application .

The purpose of step (b) is to reduce the amount of suspended substances or solids in the enzyme concentrate, to a level that is as low as possible. In preferred methods, not more than 1% by volume, preferably not more than 0.7% by volume, particularly preferably not more than 0.5% by volume of a solid are obtained in the concentrated enzyme solution by way of step (b). This can be regulated via the conventional control of the apparatus in question; in particular, the aforementioned separators yield appropriately advantageous solutions .

The core of the method of the invention, with step (c), constitutes the strongly basic anion exchange chromatography. The success of the method is therefore critically dependent on the nature of the chromatography material selected and on the experimental regime, as for example the sample collection. As already observed, the colored accompaniments here in particular are to be adsorbed onto the material, whereas the proteins, which are positively charged under conditions to be selected correspondingly, are not bound on the resin. The invention is therefore based on a strongly basic anion exchanger. In view of the fact that the majority of natural, water-soluble proteins, especially secreted proteins, tend to be water-soluble at moderate pH levels, it is particularly advantageous if the strongly basic anion exchanger in step (c) has maximum exchange capacity in the pH range of 5 to 9, preferably of 6 to 8.

Alkaline proteins in particular, such as the enzymes secreted by alkaliphilic microorganisms, for example, more particular proteases, have an isoelectric point which is situated in the alkali range, are therefore positively charged in the preferred pH range, and hence do not bind to the material in question. As mentioned, it is necessary for each protein to determine experimentally a pH that is ideal for this method, and to bring about that pH with respect to this step (c) . In examples 1 and 2, these figures, for the alkaline protease selected and the a-amylase selected, were about 7.5 and 7.

Having emerged as being particularly suitable on account of chemical properties for step (c) are strongly basic anion exchangers which have as functional groups quaternary ammonium groups, preferably those substituted with at least two alkyl groups, more preferably with at least two alkyl groups having one or two carbon atoms, and which optionally also carry a hydroxy alkyl group having one or two carbon atoms.

This requirement is met in particular by strongly basic anion exchangers having trimethylammonium or dimethylethanolammonium as functional groups. The latter is somewhat more weakly basic than the former, and so, in particular, the method can be optimized for corresponding proteins by way of this variation.

Corresponding chromatography materials characterize embodiments that are preferred accordingly.

Another characteristic of chromatography materials is their exchange capacity, which is expressed in mole equivalent per unit volume. It indicates how densely the material is populated with the functional groups. Having emerged as particularly suitable are methods wherein the strongly basic anion exchanger in step (c) has an exchange capacity of 0.7 to 1.2 meq/ml, preferably of 0.8 to 1.1 meq/ml, and more preferably of 0.9 to 1.0 meq/ml.

Another criterion influencing the separation performance of the chromatography column is the effective pore size. It must be set at a level such that retention is sufficient but at the same time the substances washed through, especially the proteins, are not excessively held back, and especially do not block the material. For both of the globular proteins investigated in the specified examples, B. lentus alkaline protease and α-amylase from Bacillus sp. A 7-7 (DSM 12368), the molecular weights of which are around 27 kD and around 58 kD respectively, it has emerged that a chromatography material can be used which has an effective pore size of 0.45 mm. For significantly larger or smaller proteins, chromatography materials with effective pore sizes that are larger or smaller accordingly ought to be selected.

In preferred methods, therefore, the strongly basic anion exchanger has effective pore sizes of 0.2 to 0.7 mm, preferably of 0.3 to 0.6 mm, more preferably of 0.4 to 0.5 mm.

Suitable in principle as supports for strongly basic anion exchangers for use in accordance with the invention are all materials described for this purpose in the prior art, including, for example, supports in gel form. Conversely, on account of their technical properties, preferred strongly basic anion exchangers in step (c) are those which are based on a porous synthetic polymer. Those based on a styrene-DVB copolymer have proven particularly advantageous.

Chromatography materials having the properties just discussed are described comprehensively in the prior art. Those from the DIAION® series are described in the handbook "DIAION®. Manual of ion exchange resins and synthetic adsorbent, Volume I" by Mitsubishi Kasei Corp. (Tokyo, Japan), June 1995, on pages 104 to 108 and in "Product Line Brochure DIAION®", June 1, 2001 version, on pages 4 to 6, available from the manufacturer, or from Summit Chemicals Europe GmbH, Dusseldorf, Germany. Strongly basic anion exchangers described therein are the DIAION®SA, DIAI0N®PA, and DIAION®HPA series. A representative of these, namely DIAION®PA30 8L, was used successfully in the examples for the present patent application.

Chemically similar materials which can be used for the chromatography in accordance with step (c) can be produced by appropriate skilled personnel in line with the details given above concerning preferred properties, or else are available from other commercial producers, and also characterize preferred embodiments. Comparable results are achieved, for example, with the DOW MSA Marathon® materials from Dow Chemicals and Amberlite®900CL materials from Rohm & Haas.

For the implementation of the chromatography in step (c) it is advantageous to observe certain conditions. These include, in particular, a particular bed volume, this being the volume ratio of the substance applied to that of the column, and a certain residence time, which is indicated favorably by way of the enzyme solution.

It has emerged as being particularly advantageous, and, correspondingly, characterizes preferred embodiments, to carry out step (c) using a bed volume of 1 to 10, preferably of 1.5 to 7, particularly preferably 2 to 4. This is the optimum, determined experimentally in accordance with the examples, in order to keep the filtrate as pure as possible and at the same time as concentrated as possible.

Suitable mean residence times for step (c) , which, correspondingly, characterize preferred embodiments, have been found to be times of 0.01 to 0.2 g, preferably 0.025 to 0.1 g, particularly preferably 0.04 to 0.06 g and especially preferably 0.05 g of enzyme per g of support material and minute . A characteristic of particularly suitable methods is that they are largely automatically controlled. One such control facility which is particularly easy to install is based on the determination of the conductivity (measurable as ps/cm) of a material for processing, at critical locations, and its use for the control. This can be done, for example, after the chromatography and can be utilized in order to separate the fractions containing substance of value (good product) from the rest. In one preferred embodiment, therefore, in a method according to the invention, step (c), in particular the separation of pre-fraction and good product and/or good product and post-fraction, is controlled by means of the conductivity of the eluate.

For boosting yields, a proposal was made as early as in patent application WO 01/37628 A2 to use filtrate for an additional washing step. Preferably, therefore, in methods according to the present invention as well, step (c) is performed by recirculating at least a portion of the pre-fraction and/or post-fraction of the ion exchange chromatography. By this means, an additional enrichment of the fraction in question is achieved with those enzyme molecules which toward the end of the peak, have not yet been washed out of the column. This step is limited in principle by the possible impurities which are possibly washed out from the column. In each individual case, the achievable concentration must be weighed against product quality, in other words purity.

For reasons of efficiency, operation during the procedure described so far has been carried out with a maximum enzyme concentration. But concentrated enzyme solutions are often not needed at such high concentrations for their technical purpose. In correspondingly preferred methods of the invention, therefore, a lower concentration is established subsequent to step (c) by dilution in a step (d) . For this purpose, mixers are employed that are known from the prior art, especially mixers which can be integrated into a continuous system.

Moreover, all liquid enzymes exhibit a tendency during storage to undergo denaturation and thereby to lose their activity. This applies in particular to proteases which hydrolyze other enzyme molecules. In one preferred method of the invention, therefore, subsequent to step (c) , a stabilizer or a stabilizer mixture is added. Such compounds are known per se from the prior art. They are, for example, stabilizers which exert their stabilizing effect via regulation of the water activity biophysically, as for example in relation to temperature fluctuations, such as polyols, or those which reversibly inactivate proteases or constitute protection against oxidation.

The stabilizer, or the stabilizer mixture, may be added optionally before, after or at the same time as a dilution (d) . It is particularly advantageous if this very step (d) is utilized in order to add a stabilizer solution together with the diluting solution, or to add a solution which exerts both effects .

The stabilizer added preferably comprises liquid compounds having hydroxyl groups, as for example a polyol such as glycerol, or, more preferably, propane-1,2-diol. Liquid compounds of these kinds may also comprise mixtures with water and/or with further stabilizing compounds.

For the development of the stabilizing effect it has emerged as being particularly favorable if the polyol stabilizer mixture is added in an amount in the range of 40 to 70% by volume, preferably 45 to 65% by volume, particularly preferably 50 to 60% by volume of the final volume.

At this point, optionally, if dilution would produce an insufficiently concentrated solution, it is possible to insert a concentration step between steps (c) and (d) , before the solution is passed through the mixer. Possible for use for this purpose in principle are all of the techniques known from the prior art, preferably those described above.

This is also another argument for operating with a very highly concentrated enzyme solution during the method so far, in order, via this massive dilution effect, to obtain a liquid enzyme which is still highly concentrated enough for the intended technical fields of use.

This dilution step may likewise be utilized in order to adjust the end product of the method to a dry substance content of 2 to 15% by weight, preferably 5 to 13% by weight, particularly preferably 8 to 12% by weight. It may likewise serve to adjust the end product of the method to a viscosity of 1 to 20 mPas, preferably 1 to 15 mPas, particularly preferably 1 to 10 mPas at 25°C, and/or to adjust the end product of the method to a sediment fraction of less than 1% by volume, preferably less than 0.75% by volume, particularly preferably less than 0.5% by volume. These values generally correlate with one another and have emerged as being particularly suitable for further storage and/or handling of liquid enzymes. As observed above, this adjustment is to be taken into account subsequent to the chromatography, or before the admixing of a stabilizer. Methods of the invention which meet these provisos are correspondingly preferred.

The invention refers to enzymes whose concentrated solutions are refined by methods according to the invention. Enzymes constitute preferred embodiments because on the one hand they are of particular technical interest and on the other hand they can be detected via their specific activities, this being possible for utilization especially for determining the optimum operating range in accordance with Examples 1 and 2 and Figure 2. Nevertheless, this method can be applied to all water-soluble proteins, provided it is possible to find their corresponding solvent systems and chromatography materials and to establish their corresponding detection reactions. This applies, for example, to peptides, such as peptide hormones, oligopeptides with pharmacological significance, or to antibodies. Antibodies would also be appropriate, for example, for detection. All of these proteins are to be understood as enzymes for the purposes of the present invention.

At the focal point of interest, however, are enzymes which can be used technically, in the conventional sense. These are, preferably, a hydrolase or an oxidoreductase, particularly preferably a protease, amylase, cellulase, hemicellulase, lipase, cutinase or a peroxidase. Methods of the invention designed in particular, via the determination and establishment of appropriate operating conditions (see above), for the processing of such enzyme solutions represent correspondingly preferred embodiments of the present invention .

Of particularly great interest, especially for the production of laundry detergents and other cleaning products, are proteases, preferably alkaline proteases, since these are particularly active and can be incorporated into alkaline formulas. Methods preferred accordingly are therefore those which are characterized by such proteases.

Preferred among these, on account of the biochemical properties of the alkaline proteases mentioned, are those methods wherein the method, in particular in step (c) , is carried out at a pH of 5 to 9, 6 to 8.5, particularly preferably 7 to 8. A protease of this kind has also been investigated in Examples 1 and 3. There it has been shown that compliance with a critical activity value is necessary for implementing an operation of the invention, in order to minimize the extent of solids which occur. Methods preferred accordingly for refining the stated concentrated protease solutions are therefore those wherein the product from step (a) is adjusted to an activity of 600 000 to 900 000, preferably 650 000 to 850 000, particularly preferably 700 000 to 800 000 HPE per g. For this regulation, in other words concentration or, optionally, dilution, the parameters known from the prior art in the component steps set out above are suitable.

In accordance with what has been said above, the end products envisaged for further use advantageously have lower activities than the kind which can be brought about in particular by dilution using stabilizing solutions. Relative to proteases, methods which constitute preferred embodiments are those wherein the end product is adjusted to an activity of 150 000 to 500 000, preferably 175 000 to 300 000, particularly preferably 200 000 to 260 000 HPE per g.

Another type of enzyme of technical significance are the a-amylases. They are used, for example, in the food industry, for the production of bakery products, or are added to laundry detergents and other cleaning products on the basis of their starch-hydrolyzing activity. In accordance with what was said for the proteases, alkaline α-amylases are particularly popular. Methods preferred correspondingly, therefore, are those which are designed for the work-up of α-amylases and are characterized by these; preferably for and/or by, those having an alkaline pH optimum.

As set out in examples 2 and 3, in preferred methods of the invention for the processing of α-amylases, the product from step (a) is adjusted to an activity of 30 000 to 50 000 TAU per g, preferably 35 000 to 45 000 TAU per g.

Since α-amylases as well are to be used usually at correspondingly lower concentrations and, moreover, are likewise to be stabilized, methods of the invention that are also preferred are those wherein the end product is adjusted to an activity of 4000 to 14 000, preferably 6000 to 12 000, particularly preferably 8000 to 10 000 TAU per g. A technical type of enzyme which is also particularly important comprises cellulases, which are used, for example, in the laundry detergent industry and in textile manufacture for the surface treatment of textiles. Accordingly, methods characterized by a cellulase, preferably having an alkaline pH optimum, also constitute correspondingly preferred embodiments of the present invention.

All of the method parameters set out so far are reflected in the respective method products - with regard, for example, to the nature of the enzyme, the degree of purity, the nature of the compounds removed, and the activities or stabilities obtained. Also preferred correspondingly, are the products obtained by way of these methods according to the invention. A subject of the invention, as well as the method, therefore, are concentrated enzyme solutions obtained by a method as described above. A further subject of the invention are compositions which comprise an enzyme which has been obtained as an intermediate, as a concentrated enzyme solution, in accordance with the second subject of the invention. Hence it is possible, for example, to use the inventively obtained concentrated enzyme solutions not in liquid form, but instead to convert them into a dry, high-purity form. This can be done, for example, by lyophilization or by incorporation into solid granules. Methods for this purpose are comprehensively described in the prior art. In this form, they can be stored over a long time or incorporated into other solid compositions, such as into solid laundry detergents and other cleaning products, for example.

This subject of the invention therefore also includes all conceivable types of cleaning product, not only concentrates but also compositions for use in undiluted form, for use on the commercial scale, in a washing machine or in laundering by hand, and/or cleaning by hand. Examples of this include detergents for textiles, carpets, or natural fibers, for which the designation "laundry detergents" is used according to the present invention. Also included, for example, are dishwashing compositions for machine dishwashers, or manual dishwashing compositions or cleaning products for hard surfaces such as metal, glass, porcelain, ceramic, tiles, stone, painted surfaces, plastics, wood or leather; for compositions of these kinds, the terms "cleaning products" is used in accordance with the present invention. Any kind of laundry detergent or cleaning product constitutes an embodiment of the present invention, in so far as it is enhanced with an enzyme which has been refined according to the method of the invention and further processed in accordance with this subject of the invention.

Embodiments of the present invention embrace all laundry detergents or cleaning products established in accordance with the prior art, and/or all appropriate forms of presentation of the laundry detergents or cleaning products of the invention. Included here in particular are solid compositions in powder form, optionally including those composed of a plurality of phases, in compressed or uncompressed form; further examples include the following: extrudates, granules, tablets or pouches, both in large containers and packaged in portions. Liquid, pasty or gel-form embodiments are included as well, in so far as, for these forms, the enzyme worked up in accordance with the invention is introduced into a further-processed form.

In one preferred embodiment, the laundry detergents or cleaning products of the invention comprise active enzymes in an amount of 2 pg to 20 mg, preferably of 5 pg to 17.5 mg, more preferably of 20 pg to 15 mg, very preferably of 50 pg to 10 mg per gram of the composition.

Besides the inventively prepared enzyme and possibly further enzymes, a laundry detergent or cleaning product of the invention optionally comprises further ingredients such as, for example, enzyme stabilizers, surfactants, examples being nonionic, anionic and/or amphoteric surfactants, bleaches, bleach activators, bleaching catalysts, builders, solvents, thickeners, and optionally, as further customary ingredients, sequestrants, electrolytes, optical brighteners, graying inhibitors, color transfer inhibitors, foam inhibitors, colorants and/or fragrances, active antimicrobial ingredients and/or UV absorbers, to list only the most important classes of substance. Corresponding formulas are described comprehensively in the prior art.

Conversely, on account of the advantageous properties of the liquid enzymes obtained in accordance with the invention, more particularly on account of their clear appearance and the fact that they can be used without further work-up, preferred compositions are those which comprise a correspondingly concentrated enzyme solution. Of particular interest in this context are those compositions which are present overall in a liquid, pasty or gel form. They can be easily metered, comprise the enzyme in the desired activity, and have an appealing appearance, at least as far as the enzyme component is concerned. This was set out at the outset as being desirable .

This is true especially in respect of laundry detergents and cleaning products designed for the end user. In a particularly preferred form, therefore, the compositions of this subject of the invention are laundry detergents or are cleaning products. These fall within the definition given above and may comprise the substances indicated there. Furthermore, in accordance with this subject of the invention, they possess overall a liquid, gel or pasty form, into which the refined products according to the invention can be incorporated easily, in other words by techniques which are known per se.

Examples

Example 1

Refinement of a concentrated protease solution Removal of the biomass

After the fermentative production, as described in WO 91/02792 Al, of the protease substance of value, the biomass was separated off virtually to completion by way of the known techniques of separation, microfiltration, and sterile filtration. This was followed, as described in patent application WO 01/37628 A2, by concentration, in other words removal of the solvent by an ultrafiltration until the protease concentrate had an activity of 300 000 to 400 000 HPE per g, as determinable by the method of van Raay, Saran, and Verbeek, in accordance with the publication "Zur Bestimmung der proteolytischen Aktivitat in Enzymkonzentraten und enzymhaltigen Wasch-, Spul- und Reinigungsmitteln" in Tenside (1970), Volume Ί_, pp. 125-132. Additionally, a pH of 7.5 was set using a 30% strength CaCl2 solution. The solution had a solids fraction of less than 1% by volume, as determinable by means of a bench or laboratory centrifuge by centrifugation for 10 min at 7000 g. The protease present also exhibited a positive charge at an ionic strength of 1 to 20 mS/cm.

Determination of the optimum operating range

Samples of the solution obtained after removal of the biomass were taken during the ultrafiltration (values up to 400 000 HPE), and, as described below, were concentrated further via a separator, with determination of the solids fraction as a function of the respective activity, as indicated above. The activity measurements took place in each case at a pH of 7.5, at a temperature of 20°C, and at an ionic strength of 10 mS/cm. This gave the dependency relationship shown in table 1 and in figure 2.

Table 1: Dependency of solids fraction on concentration of active protease_ _

As can be seen from this, the solids fraction of a concentrated protease solution rises disproportionately above about 900 000 HPE/g, and so the range from about 700 000 to 800 000 HPE/g may be regarded as the optimum operating range with maximum activity in conjunction with minimum solids fraction.

Step (a) : Concentration of the enzyme solution to the operating range

The enzyme solution ultimately obtained in the preceding step by ultrafiltration was concentrated, on the basis of this measurement outcome, to a figure of 800 000 HPE/g, using a thin film evaporator. The conditions observed here were as follows: product temperature above 35°C, vacuum about 20 mbar, largely constant pH about 7.5, and minimization of the solids fraction of the enzyme concentrate of less than 3% by volume, to minimize the losses in the subsequent step.

Step (b): Separation of the precipitates formed (solids)

The solids formed by concentration (precipitates) were removed by mechanical separation, based on the principle of gravitational or centrifugal separation. Specifically, to remove the solids, a separator with intermittent sediment discharge was used, this being the BTPX 205 device from ALFA LAVAL, with a clarifying area of 11 700 m2, operated with a g of 12 800 and with a throughput of 200 1/h. The result was a largely solids-free enzyme concentrate with a solids content of less than 0.2% by volume, determined as indicated above. The activity was still about 800 000 HPE/g.

Step (c): Strongly basic anion exchange chromatography

The chromatographic decolorizing took place in a fixed bed using the strongly basic anion exchanger DIAION® Pa 308L, available from Mitsubishi, Tokyo, Japan, or from Mitsubishi Chemical Europe GmbH, Dusseldorf, Germany. The quality of decolorizing and hence the stability of the enzyme were controlled via the bed volume ratio (BM; volume ratio of the enzyme concentrate to the resin) and via the residence time; specifically, a ratio of 2 to 5 BM and an amount 0.05 kg of enzyme solution per kg of resin per min were set. The principle is based on the fact that the enzyme is repelled by the support material, and hence is carried along in the stream of liquid, while the colorants are bound on the immobile support. Some of the post-fraction was passed through the column again in order to increase the yield. Subsequently, by rinsing with NaCl solutions and NaOH solutions, the compounds adsorbed to the column, especially the colorants, were washed out, and the fixed bed was regenerated in this way.

Step (d) : Mixture, stabilization and activity adjustment with solvent

The filtrate with an activity of around 700 000 HPE per g was mixed directly, in other words on line in a static mixer, with propane-1,2-diol, which at the same time exhibits a stabilizing effect. A solvent fraction of around 55% by volume was taken.

The properties of the liquid enzyme refined over these four steps were as follows, with the activity being determined as defined above and a determination made according to the internationally customary CIE color scale, defined in DIN 5033-3 and DIN 6174:

Activity 260 000 HPE/g

Color: L > 96 b* < 14

Viscosity: < 10 mPas pH: about 7

The liquid obtained was virtually clear and exhibited no subsequent precipitation immediately after the method. The further stability is described in example 3.

Example 2

Refinement of a concentrated amylase solution

An amylase solution refined in accordance with the invention was produced as in example 1, except for the following differences. A fermenter batch was used which contained, as substance of value, the cx-amylase described in patent application WO 02/010356 A2. Since the α-amylase has a different isoelectric point from the protease, a pH of 6.25 was set even before step (a), and throughout the entire method a pH of 6 to 6.5 was maintained, in order to keep the charge on the amylase positive.

Activity determination

In order to determine the amylolytic activity in TAU, a modified p-nitrophenylmaltoheptaoside is used whose terminal glucose unit is blocked by a benzylidene group, which is cleaved by amylase to give free p-nitrophenyloligosaccharide, which in turn is converted by means of the auxiliary enzymes glucoamylase and alpha-glucosidase to form glucose and p-nitrophenol. The amount of p-nitrophenol released is therefore proportional to the amylase activity. Measurement takes place, for example, using the Quick-Start® assay kit from Abbott, Abott Park, Illinois, USA. The increase in absorption (405 nm) in the assay mixture is detected by photometry at 37°C over 3 minutes against a blank. Calibration takes place via an enzyme standard with known activity (for example,

Maxamyl®/Purastar® 2900 from Genencor, Palo Alto, CA, USA, at 2900 TAU/g). Evaluation takes place by clotting of the absorption difference dE (405 nm) per min against the enzyme concentration of the standard.

Determination of the optimum operating range

During the ultrafiltration and the subseguent separation, samples of different concentration were taken as in example 1, and likewise the solids fraction was determined as a function of the respective activity. The measurements were made in each case at a pH of 6.25, at a temperature of 20°C, and at an ionic strength of 10 mS/cm. This gave the dependency relationship shown in table 2 and in figure 2.

Table 2: Dependency of solids fraction on concentration of active a-amylase

As can be seen from this, for the amylase, there is a dependency relationship between solids fraction and the concentration of active enzyme that is comparable with that of the protease (see above) ; this is indicated in figure 2 in 100 TAU per g. Accordingly, the solids fraction of a concentrated amylase solution rises disproportionately above about 50 000 TAU/g, which means that the range from about 35 000 to 45 000 TAU/g can be regarded as the optimum operating range with maximum activity in conjunction with minimum solids fraction. In accordance with the invention, therefore, operation ought to take place below this range.

Through step (a), carried out in analogy to example 1, therefore, an activity of 35 000 to 45 000 TAU per g was established, and the further method was carried out as in example 1, in other words also on the same anion exchange chromatography material. In step (d), in an analogous way, a concentration of 9000 TAU per g was established by mixing with propane-1,2-diol.

The properties of the liquid enzyme refined over these steps were as follows:

Activity 9000 TAU/g pH: about 6.25

The liquid was virtually clear and, like the protease in example 1, exhibited no precipitation immediately after the method.

Example 3

Storage stability of the protease in a liquid laundry detergent matrix

In order to determine the storage stability of a protease refined in accordance with the invention, in comparison to the nonpurified enzyme and to a commercial product, in each case in the same matrix comprising liquid laundry detergent, the following three samples were prepared: (1.) an unrefined protease, as present in accordance with example 1 after ultrafiltration and before step (a); (2.) the fully purified commercial product Savinase®l6.0 LEX, available from Novozymes, Bagsvaerd, Denmark; and (3.) the protease refined according to example 1. All three samples were introduced in an activity of 260 000 HPE per g in a solution of 55% by volume of propane-1,2-diol in water, and were incorporated in a fraction of 0.4% by volume into a liquid laundry detergent matrix of customary composition :

On the CIE color scale (see above), the protease samples introduced into this matrix gave L values of (1.) 78, (2.) 99, and (3.) 97, respectively; in other words, the inventively refined protease was almost as clear as the fully purified product. Samples were taken at regular intervals over a period of 12 weeks and, as indicated above, the residual activities were ascertained. This produced the values indicated in table 3, and shown graphically in figure 3.

Table 3: Storage stability of the protease in a liquid laundry detergent matrix

The figure reported in each case is the residual HPE activity in %.

It is seen that the inventively refined protease combines virtually the same color value as the fully purified product with a significantly greater stability than the latter, and that the inventively refined protease loses only little activity relative to the dark, non-purified enzyme in a laundry detergent matrix.

Description of the figures

Figure 1: Block flow diagram of the inventive refinement of concentrated enzyme solutions Depicted are the following steps: (a) concentration of the enzyme solution to the operating range, with supernatant solution being taken off; (a') optional deodorization; (b) separation of the resultant precipitates (solids); (c) strongly basic anion exchange chromatography, in which the compounds adsorbed to the column, more particularly colorants, are washed out by rinsing with corresponding media in separate steps, and the column is regenerated; and (d) stabilization and activity adjustment by admixing of a solvent.

Figure 2: Dependency relationship between the solids fraction and the concentration of active enzyme, determined in examples 1 and 2

The solids fraction is reported in % by volume, as determinable using a laboratory centrifuge via centrifugation for 10 min at 7000 g. The activity of the protease is reported in 1000 HPE/g, and that of the α-amylase in 100 TAU/g. The operating ranges emphasized are those considered to be optimum in accordance with the present patent application.

Figure 3: Storage stability of the protease in a liquid laundry detergent matrix, determined in example 3 Determined for: 1. unrefined protease; 2. the commercial product Savinase®l6.0 LEX from Novozymes; and 3. protease refined according to example 1.

Claims

A process for refining concentrated enzyme solutions containing colored Maillard compounds comprising the following steps: (a) preparing a concentrated enzyme solution, (b) separating solids, in particular of foreign proteins and / or inactive enzymes, and (c) separating the colored Maillard compounds by adsorption on a highly basic anion exchanger, the highly basic anion exchanger having quaternary ammonium groups as functional groups, having an exchange capacity of 0.7 to 1.2 meq / ml, having an effective pore size of 0.2 to 0.7 mm and is based on a porous plastic polymer as the carrier, the highly basic anion exchanger having a pH range of 5 to 9 having maximum exchange capacity, characterized in that in the concentrated enzyme solution by step (b) is more than 1 vol.% solids and the enzyme proteins throughout the process remain in solution.

Process according to claim 1, characterized in that an ultrafiltration concentrate is introduced in step (a).

Method according to claim 1 or 2, characterized in that step (a) is carried out by means of a thin layer evaporator.

Process according to one of claims 1 to 3, characterized in that an enzyme concentrate containing no more than 4 to 20% by weight, preferably no more than 4.5 to 15% by weight, is prepared by step (a). %, especially preferably no more than 5 to 10% by weight of dry matter.

Process according to one of claims 1 to 4, characterized in that in step (a) with step (a ') a deodorization of the concentrated enzyme solution is carried out.

Process according to one of claims 1 to 5, characterized in that step (b) is carried out by a mechanical separation method, preferably due to a gravitational or centrifugal separation.

Method according to claim 6, characterized in that step (b) is carried out with a separator, preferably a continuously operating separator, especially preferably a discontinuous sample discharge separator.

Process according to one of claims 1 to 7, characterized in that in the concentrated enzyme solution by step (b) no more than 1% by volume, preferably not more than 0.7% by volume, in particular, is obtained. preferably not more than 0.5 vol.% solids.

Process according to one of claims 1 to 8, characterized in that the highly basic anion exchanger for step (c) in the pH range of 5 to 9, preferably from 6 to 8, has maximum exchange capacity.

Process according to one of claims 1 to 9, characterized in that the highly basic anion exchanger for step (c) as functional groups has quaternary ammonium groups substituted with at least two alkyl groups, preferably with at least two alkyl groups with 1 or 2 carbon atoms. , optionally, further, having a hydroxyalkyl group having 1 or 2 carbon atoms.

Process according to claim 10, characterized in that the highly basic anion exchanger for step (c) as functional groups has trimethyl-ammonium or dimethylethanol-ammonium groups.

Process according to one of claims 1 to 11, characterized in that the highly basic anion exchanger for step (c) has a exchange capacity of 0.8 to 1.1 meg / ml, preferably 0.9 to 1.0 meg / ml. .

Process according to one of claims 1 to 12, characterized in that the highly basic anion exchanger for step (c) has effective pore sizes of 0.3 to 0.6 mm, preferably 0.4 to 0.5 mm.

Process according to one of claims 1 to 12, characterized in that the highly basic anion exchanger for step (c) depends on a styrene-DVB copolymer.

Method according to one of claims 1 to 14, characterized in that step (c) is carried out with a bed volume of 1 to 10, preferably 1.5 to 7, especially preferably 2 to 4.

Process according to one of claims 1 to 15, characterized in that step (c) takes place at an average residence time of 0.01 to 0.2 g, preferably 0.025 to 0.1 g, especially preferably 0.04 to 0, 06 g and particularly preferred at 0.05 g of enzyme per ml. g of carrier material and minute.

Process according to one of claims 1 to 16, characterized in that step (c), in particular the separation between the process product and the material product and / or the material product and the after product, is controlled via the conductivity of the eluate.

Process according to one of claims 1 to 17, characterized in that step (c) is carried out during the return of at least part of the precursor and / or after-product of the ion exchange chromatography.

Process according to one of claims 1 to 18, characterized in that a lower concentration value is set in connection with step (c) in a step (d).

Method according to one of claims 1 to 19, characterized in that, in addition to step (c), if necessary before, after or simultaneously with a dilution according to claim 19, a stabilizer is added, preferably simultaneously with step (d). .

Process according to claim 20, characterized in that it is a polyol, preferably 1,2-propanediol, in connection with the stabilizer.

Method according to claim 21, characterized in that the stabilizer is added in an amount range of 40 to 70 vol.%, Preferably of 45 to 65 vol.%, Especially preferably 50 to 60 vol.% Of the final volume.

Process according to one of claims 19 to 22, characterized in that the process final product is adjusted to a dry matter content of 2 to 15% by weight, preferably 5 to 13% by weight, especially preferably of 8 to 12% by weight.

Process according to one of claims 19 to 23, characterized in that the process final product is set to a viscosity value of 0.001 to 0.02 Ns / m 2 (1 to 15 mPas), preferably 0.001 to 0.015 Ns / m 2 (1 to 15 mPas). particularly preferred at 0.001 to 0.01 Ns / m 2 (1 to 10 mPas) at 25 ° C.

Process according to one of claims 19 to 24, characterized in that the process final product is adjusted to a sediment content of less than 1% by volume, preferably less than 0.75% by volume, particularly preferably less than 0.5% by volume. %.

Process according to one of Claims 1 to 25, characterized in that it is a technically useful enzyme, preferably a hydrolase or an oxidoreductase, especially preferably a protease, amylase, cellulase, hemicellulase, lipase, cutinase or a peroxidase.

Process according to claim 26, characterized in that it is a protease, preferably an alkaline protease.

Process according to claim 27, characterized in that it is carried out in particular in step (c) at a pH of 5 to 9, preferably 6 to 8.5, especially preferably 7 to 8.

Process according to claim 27 or 28, characterized in that the product of step (a) is set to an activity value of 600,000 to 900,000, preferably 650,000 to 850,000, particularly preferably 700,000 to 800,000 HPE per unit. g.

Process according to one of claims 22 to 24, characterized in that the final product is set to an activity value of 150,000 to 500,000, preferably 175,000 to 300,000, especially preferably 200,000 to 260,000 HPE per unit. g.

Process according to claim 26, characterized in that it is an α-amylase, preferably with an alkaline pH optimum.

Method according to claim 26 or 31, characterized in that the product of step (a) is set to an activity value of 30,000 to 50,000 TAU per day. g, preferably 35,000 to 45,000 TAU per g.

Process according to one of claims 31 to 32, characterized in that the final product is set to an activity value of 4,000 to 14,000, preferably 6,000 to 12,000, especially preferably 8,000 to 10,000 TAU per day. g.

Process according to claim 26, characterized in that it is a cellulase, preferably with an alkaline pH optimum.